JCM Accepted Manuscript Posted Online 27 January 2016 J. Clin. Microbiol. doi:10.1128/JCM.02605-15 Copyright © 2016 Kakumanu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
1
Development and Validation of an Improved PCR Method Using 23S-5S Intergenic Spacer for
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Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and
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Laboratory Animals
4 5 6 7
Madhavi L. Kakumanua, Loganathan Ponnusamya, Haley T. Suttona, Steven R. Meshnickb,
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William L. Nicholsonc, and Charles S. Appersona,d,#
9 10 11
Department of Entomology, North Carolina State University, Raleigh, North Carolina, USAa;
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Department of Epidemiology, Gillings School of Global Public Health, University of North
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Carolina, Chapel Hill, North Carolina, USAb; Rickettsial Zoonoses Branch, National Center for
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Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention,
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Atlanta, Georgia, USAc; Center for Comparative Medicine and Translational Research, North
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Carolina State University, Raleigh, North Carolina, USAd
17 18 19
Running head: Improved PCR assay for detection of Rickettsia spp.
20 21 22
#
Address correspondence to Charles S. Apperson,
[email protected]
23
ABSTRACT
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A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue
25
samples from humans and laboratory animals. Primers were designed for the nested run
26
to amplify a variable region of the 23S-5S intergenic spacer of Rickettsia spp. The newly
27
designed primers were evaluated using genomic DNA from 11 Rickettsia species,
28
belonging to the spotted fever, typhus, and ancestral groups and in parallel compared to
29
other Rickettsia-specific PCR targets (ompA, gltA and 17-kDa). The new 23S-5S nested
30
PCR assay amplified all 11 Rickettsia spp. but the assays employing other PCR targets
31
did not. The novel nested assay was sensitive enough to detect one copy of a cloned
32
23S-5S IGS fragment from “Candidatus R. amblyommii”. Subsequently, the detection
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efficiency of the 23S-5S nested assay was compared to the other three assays using
34
genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S
35
assay detected Rickettsia DNA in 45% of the ticks while the amplification rate of the three
36
other assays ranged between 5-20%. The novel PCR assay was validated using clinical
37
samples from humans and laboratory animals that were known to be infected with
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pathogenic species of Rickettsia. The nested 23S-5S PCR assay was coupled with
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reverse line blot hybridization with species-specific probes for high-throughput detection
40
and simultaneous identification of the species of Rickettsia in the ticks. Rickettsia
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amblyommii, R. montanensis, R. felis and R. bellii were frequently identified species
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along with some potentially novel Rickettsia that were closely related to R. bellii and R.
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conorii.
44
2
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Rickettsia species are Gram-negative, obligate intracellular bacteria vectored by a diverse
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array of arthropods (1). These bacteria are broadly categorized into the spotted fever group
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(SFG), typhus group (TG) and two ancestral groups of rickettsiae (2). Rickettsiae are
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noteworthy because of their global distribution and large array of species identified as human
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pathogens (3). Ticks are the principle vectors of SFG rickettsiae, and in particular, Dermacentor
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variabilis Say, the American dog tick, is an established vector of R. rickettsii, the causative
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organism of Rocky Mountain spotted fever (RMSF). Reported cases of SFG rickettsioses
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(including RMSF) have escalated in the United States in recent years with a concomitant
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decrease in the case fatality rate (4). Consequently, the increase in reported cases of illness
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could be caused by less virulent strains of R. rickettsii (5, 6). However, recent studies of
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rickettsiae prevalence in D. variabilis have either failed to detect R. rickettsii (7-11) or found it to
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be exceedingly rare (12, 13). Accordingly, it is possible that mildly pathogenic Rickettsia, some
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of which may be novel species (4, 14-16), are responsible for the escalation of reported cases.
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To understand the role of novel pathogens in driving the recent increase in SFG rickettsioses,
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investigations of the diversity of Rickettsia species in D. variabilis ticks should be carried out
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(17).
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PCR based assays are commonly used for surveillance of rickettsial pathogens in field-
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collected ticks (18). The 17-kDa protein gene, ompA, ompB, SCA4 and gltA are some of
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commonly targeted genes for detection of Rickettsia spp. infection in ticks (8, 13, 19-21).
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Recently, the 23S-5S intergenic spacer (IGS) has been reported to be a robust target for
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detection of Rickettsia (22-24). The 23S-5S IGS is a noncoding region of DNA that is present in
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Rickettsia species (25, 26). The 23S-5S IGS has conserved ends and hypervariable central
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regions exhibiting sequence diversity, making it an additional target for identification of
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Rickettsia DNA to the species level (22). While PCR assays targeting different regions of the
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Rickettsia genome have been used successfully to detect rickettsiae in environmental samples,
3
70
inconsistencies in the amplification rate of different gene targets in the same samples is not
71
uncommon (8, 23). This problem could potentially result from a lack of specificity of the primers
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for potentially novel species or to a low copy number of the target genes.
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The objective of our study was to develop a new set of primers for a nested 23S-5S IGS
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PCR assay that would increase the efficiency of amplification of Rickettsia spp. even at low
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abundance. We then coupled the 23S-5S PCR assay with reverse line blot (RLB) hybridization
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for high-throughput screening and simultaneous identification of Rickettsia in ticks.
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MATERIALS AND METHODS
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Primer design and PCR optimization for 23S-5S IGS. A set of new primers were designed
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for nested PCR assay targeting ~350 bp DNA fragment from the 23S-5S intergenic spacer
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(IGS) region of Rickettsia. The 23S-5S sequences of some Rickettsia spp. (including SFG and
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ancestral groups) were retrieved from GenBank, aligned using Clustal W program (27). We
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used the MEGA software version 6 package (28) to identify 20-22 bp regions that were
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conserved among all groups of Rickettsia. The newly designed nested PCR primers were:
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RCK/23-5N1F (5’ TGTGGAAG CACAGTAATGTGTG 3’) and RCK/23-5N1R (5’
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TCGTGTGTTTCACTCATGCT 3’). Amplification conditions of the thermocycler were optimized
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by conducting temperature gradient PCR on genomic DNA of some known Rickettsia species
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(positive control DNA) and on tick samples that were previously identified as positive for
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rickettsiae. After optimization, assays were conducted on genomic DNA from 11 known species
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of Rickettsia belonging to SFG, typhus and ancestral groups (Table 1). The DNA for the
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Rickettsia spp. were obtained from the Rickettsial Zoonoses Branch, the Centers for Disease
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Control and Prevention (Atlanta, GA, USA), except for R. monacensis, for which cell culture
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material was obtained from Fuller Laboratories (Fullerton, CA, USA).
93 94
Primary PCR amplifications were conducted in a 20 µL reaction mixture consisting of 1 µL of genomic DNA (2 ng), 10 µL of AmpliTaq Gold® PCR Master Mix (Cat No. 4398881, Life
4
95
Technologies, USA), 1 µL of primers RCK/23-5-F (10 µM), 1 µL of primer RCK/23-5-R (10 µM)
96
(22) , and 7 µL of nuclease-free water. The nested PCR reaction mix consisted of 1 µL of
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primary amplicons as template DNA, 10 µL of AmpliTaq Gold® PCR Master Mix, 1 µL of primer
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RCK/23-5N1F (10 µM), 1 µL of primer RCK/23-5N1R (10 µM), and 7 µL of nuclease-free water.
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Biotin-modified (5’ end) nested primers were used if the amplicons were to be used in RLB
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hybridization reactions. The PCR conditions used for amplification of 23S-5S IGS fragments
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were: 95 °C for 10 min, 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 65 °C for 1.30 min, and a
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final cycle of 65 °C for 7 min (for primary reaction, modified protocol of Lee et al. (23)), and 95
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°C for 10 min, 30 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1.30 min, and a final
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cycle of 72 °C for 10 min (for nested reaction). All the primers and probes used in this study
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were ordered from Life Technologies (Grand Island, NY, USA).
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Amplification of positive control Rickettsia. We compared our nested 23S-5S IGS PCR
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assay with nested/semi-nested PCR assays employing three commonly-used genes (gltA,
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ompA, and 17-kDa) for detection of Rickettsia spp. in ticks. PCR assays were carried out using
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genomic DNA for the aforementioned 11 Rickettsia spp. A concentration of 2 ng gDNA from
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each positive control sample was used as template in primary PCR for all the PCR targets. The
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PCR reaction mix of 20 µL was comprised of 10 µL of reaction buffer, 1 µL each of forward and
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reverse primers (10 µM) with 1 µL of genomic DNA (2 ng) in primary reactions; and 1 µL of the
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amplicon from primary PCR reaction was used as the template in subsequent nested PCR
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assays. Negative controls (reaction mix with no DNA) were included in each set of PCR
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reactions. PCR primers 17-kDa (29), ompA (30, 31) and gltA (32, 33), and assay conditions
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used for the gene targets were described in Lee et al. (23). Amplicons, including 23S-5S, were
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subjected to electrophoresis on ethidium bromide stained 1.2% agarose-TAE gels and the
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banding patterns were visualized by UV transillumination. Amplicons were purified and
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subjected to DNA sequencing (Sanger) using the forward primer from the nested/semi-nested
5
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reactions. Two separate PCR assays were completed for each Rickettsia sp. for all four
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gene/IGS targets.
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DNA extraction from Dermacentor variabilis ticks. Deplete adult ticks, collected from
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field sites in North Carolina by flagging vegetation (34), were preserved in 95% ethanol upon
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collection and stored at -20 °C until processed. Genomic DNA was extracted individually from
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40 D. variabilis adult ticks, using methods previously described (35). Crude DNA samples were
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purified with the Wizard® DNA Clean-Up System (Promega, Madison, WI, USA) and the purified
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DNA was quantified with a NanoDrop® (Thermo Scientific, Wilmington, DE, USA). All 40 tick
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genomic DNA samples were normalized to a concentration of 75 ng/µL and stored at -20 °C for
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later use.
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Comparison of the amplification efficiency of PCR assays targeting Rickettsia in field-
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collected ticks. Genomic DNA samples from the 40 D. variabilis that had been normalized to
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75 ng/µL DNA were amplified for 4 Rickettsia genus-specific PCR targets (23S-5S IGS, gltA,
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ompA, 17-kDa). All the DNA samples were analyzed using the respective PCR thermo-cycling
134
conditions outlined above with two replicate (separate) assays completed for each tick/PCR
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target.
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Validation of 23S-5S nested PCR assay. Tick samples. The new nested PCR assay was
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further validated using genomic DNA from 17 pools of D. variabilis ticks, which were tested
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previously for SFG rickettsiae by PCR targeting the ompB gene (10).
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Clinical samples. In addition to ticks, three samples of gDNA from humans were tested
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using the nested 23S-5S assay as described above. These samples were from a larger set of
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samples from clinical patients who were tested for R. rickettsii infection by Kato et al. to confirm
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RMSF (36). Similarly, blind tests were conducted using genomic DNA samples from skin and
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organ tissues extirpated from laboratory animals (dogs, guinea pigs, goats, rabbits) that had
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been challenged with R. rickettsii (37) or R. slovaca (Zemtsova et al. unpublished studies).
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Also, genomic DNA from skin, blood and other organ tissue samples (lung, liver, spleen, kidney, 6
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lymph node) taken from some of the laboratory animals were extracted in our laboratory (35)
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and analyzed for Rickettsia using the new PCR assay. All the clinical samples were tested
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twice in separate PCR assays, each using 2.5 µL of template DNA in the primary cycle and 1 µl
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of the primary amplicon in the subsequent nested cycle. Zemtsova et al. originally tested the
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samples from laboratory animals that were experimentally infected with Rickettsia using a SYBR
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green-based real-time PCR with 5 μL of template DNA (37). 23S-5S IGS fragments amplified
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from clinical samples were sequenced as described below to confirm the identity of the
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Rickettsia spp.
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Quality control of PCR assays. All tick and clinical tissue samples were processed in a
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non-ventilated PCR enclosure. PCR reactions were prepared in the non-ventilated PCR
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enclosure or a laminar flow hood, which were located in separate rooms. On each occasion
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prior to being used, these work areas were thoroughly cleaned with ethanol and exposed to UV
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light. Every PCR run included positive (DNA from Rickettsia parkeri) and negative (PCR mix but
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no template DNA) control samples.
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Establishing the limits of detection. Plasmid DNA ligated with targeted PCR fragments of
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“R. amblyommii” was used to establish the limits of detection (38). Briefly, 23S-5S, ompA and
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gltA PCR fragments of “R. amblyommii” were cloned separately using the pGEM®-T Cloning Kit
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(Cat. No. A1360; Promega, Madison, WI, USA). Individual white colonies from each plate were
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picked and grown overnight at 37 oC in LB broth supplemented with ampicillin (100 µg mL-1).
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Plasmid DNA from each overnight culture was extracted using UltraClean® Standard Mini
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Plasmid Prep Kit (Cat. No.12301; Mo Bio Laboratories Inc., CA, USA). The identity of inserts
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from the plasmid DNA were confirmed by amplifying with vector primers M13F and M13R and,
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sequencing and analyzing the DNA fragments using BLASTN (39). Subsequently, primary and
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nested PCR assays of the 3 targets were conducted, using their respective protocols (as
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described above), on ten-fold serial dilutions of plasmid DNA ranging from 106 to 1 copy per µL.
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The copy number in the dilutions was calculated using the DNA copy number calculator on Life 7
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Technologies website. (https://www.lifetechnologies.com/us/en/home/brands/thermo-
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scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-
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library/thermo-scientific-web-tools/dna-copy-number-calculator.html). To determine if tick
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genomic DNA interfered with the amplification of Rickettsia DNA, a series of plasmid DNA
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dilutions each spiked with 75 ng of D. variabilis DNA that was negative for Rickettsia DNA was
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also tested concurrently.
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Reverse line blot (RLB) hybridization. RLB hybridization was conducted using ~350 bp
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23S-5S nested amplicons of Rickettsia DNA from positive controls and D. variabilis ticks as
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described by Lee et al. (23). Briefly, amplicons from 23S-5S nested PCR assays displaying the
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expected-size band on agarose electrophoresis gels were diluted by mixing 10 µL of the PCR
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product and 180 µL of 2X SSPE/0.1% SDS solution and used in RLB hybridization assay. The
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hybridization temperature was 52 °C for 1 hour and stripping solution concentration and
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temperature were 0.5% SDS and 60 °C, respectively. Hybridized PCR products were detected
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by chemiluminescence with a ChemiDoc-It™TS2 Imaging System (UVP, Upland, CA, USA),
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following incubation of the membrane in ECL detection liquid (Amersham, Little Chalfont,
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Buckinghamshire, United Kingdom).
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Sequencing and phylogenetic analyses. Nucleotide sequence analysis of the 23S-5S
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IGS fragments was used to confirm the identity of the Rickettsia spp. used as positive controls
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and to identify the Rickettsia spp. in the D. variabilis DNA samples that were analyzed by RLB
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hybridization. Attempts were made also to sequence amplified DNA from the other three PCR
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targets. Forward primers used in the nested-PCR was employed for sequencing Rickettsia spp.
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(23-5SN1F [23S-5S], 190.70p [ompA], RpCS896p [gltA]), and 17kD1F [17-kDa]), which was
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performed by Eton BioScience, Inc. (Research Triangle Park, NC, USA). DNA sequences were
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identified by blasting (BLASTN) the partial Rickettsia sequences in the NCBI database.
196 197
The 23S-5S sequences from the positive control Rickettsia spp. and ticks were aligned with sequences deposited in GenBank using Clustal X version 2.0 (27) and the sequences were 8
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trimmed at both ends to and average length of 330 bp. Phylogenetic analysis was conducted
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with the neighbor-joining method (40), using the Kimura two-parameter model with partial gap
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deletion and a cutoff of 95% site coverage. Evolutionary distance was calculated and bootstrap
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analysis with 1000 iterations was carried out with the MEGA6 software package (28).
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The 23S-5S sequences of the known Rickettsia spp. generated in this study were submitted
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to GenBank (Accession nos.: KT340601 – KT340611). The 23S-5S, gltA, and ompA sequences
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from D. variabilis ticks were also deposited in the Genbank (Accession nos.: 23S-5S: KT374186
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– KT374204; gltA: KT374205 – KT374212; ompA: KT374213 – KT374216).
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RESULTS
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Amplification of positive control Rickettsia spp. and limits of detection. Attempts were
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made to amplify four PCR targets (17-kDa, ompA, gltA and 23S-5S IGS) from 11 Rickettsia
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species, including members of the SFG, TG, R. bellii, and R. canadensis, (Table 1). The 23S-
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5S nested method amplified all Rickettsia spp., producing the expected size band of ~350 bp.
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The identity of 23S-5S nested amplicons of 11 positive controls was confirmed by sequencing
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and aligning them to known Rickettsia spp. sequences from GenBank. At the same DNA
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concentration, the PCR targeting ompA amplified all the SFG rickettsiae but not R. bellii, R.
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canadensis or R. typhi. The gltA assay amplified all but R. bellii and the 17-kDa assay did not
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produce visible bands for R. bellii and R. canadensis. Failure to amplify gltA of R. bellii appears
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to be due to miss-matches of reverse primers for the primary (4 of 22 nucleotides) and nested (3
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of 19 nucleotides) amplifications with the target gene sequence (GenBank Accession No.
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DQ146481.1).
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Amplification of serially diluted plasmid DNA ligated with 23S-5S, ompA and gltA amplicons
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of “R. amblyommii” were tested separately to compare their levels of detection. As low as 1
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copy of 23S-5S fragment in the primary PCR reaction was enough to produce a highly visible
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gel band in the nested PCR reaction. The sensitivity of ompA gene was on par with 23S-5S;
9
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however, the sensitivity of gltA gene amplification was 10-fold lower than the other two PCR
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targets. The amplification sensitivity decreased by one 10-fold dilution in the presence of tick
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DNA for all three PCR targets. Serial dilution assays for the three targets were repeated twice
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in separate PCR amplifications.
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Validation of 23S-5S IGS PCR assay. Tick samples. Seventeen D. variabilis DNA
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samples that were previously analyzed for Rickettsia ompB gene in another laboratory were
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used to validate our 23S-5S IGS nested assay. Seven out of 17 samples were reported to be
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positive for R. montanensis and 10 samples were PCR-negative. With the 23S-5S nested
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assay, all 7 positive samples and two of the 10 ompB gene negative samples produced
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expected-size bands (~350 bp) on agarose gels (data not shown). The two samples that failed
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to amplify in PCR assays targeting the ompB gene but tested positive in our 23S-5S nested
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assay were subsequently sequenced and identified as R. montanensis.
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Clinical samples. Clinical samples from humans and laboratory animals were used to
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further validate the nested 23S-5S assay. All three DNA samples from humans known to be
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infected with R. rickettsii tested positive using the nested assay based on visual detection of
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expected-sized bands on agarose electrophoresis gels. These amplicons were sequenced and
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found to be 99-100% homologous to R. rickettsii (GenBank Accession No. CP006010.1).
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Results of the blind tests of samples from laboratory animals carried out with the new nested
241
PCR assay were highly concordant with those of the real-time PCR tests. The 23S-5S nested
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assay detected Rickettsia DNA in 88% (15 of 17) gDNA samples that were reported positive
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when tested by real-time PCR. For the tissue samples extracted in our laboratory, the 23S-5S
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nested assay detected DNA of Rickettsia in 4 of 5 samples that were positive by real time-PCR.
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Notably, the nested 23S-5S assay detected Rickettsia DNA in one sample that was reported to
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be negative by real-time PCR. The majority (12 of 17) of Rickettsia-positive tissue samples
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were from skin, but skin comprised only 65% (13 of 20) of the total number of tissue samples
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tested. Rickettsia DNA was detected in skin samples resected at the site of the tick bite as well 10
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as from distally located sites. Generally, rickettsiae were not detected in organ tissue samples
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from the spleen, heart, kidney, or lung. The Rickettsia spp. in laboratory animal clinical samples
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were 99-100% homologous to partial 23S-5S IGS sequences of R. rickettsii (Accession No.
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CP00610.1) and R. slovaca (Accession No. CP003375.1) deposited in GenBank.
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Comparison of the amplification efficiency of PCR assays targeting Rickettsia in field-
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collected ticks. Attempts were made to amplify Rickettsia spp. from 40 D. variabilis DNA
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samples with the 4 PCR targets. After the primary 23S-5S amplification only 2 out of the 40
256
samples (5%) showed expected sized bands in agarose electrophoresis gels in both replications
257
(Fig. 1), but the detection rate increased to 45% (18/40) after the nested step in the assay. The
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other PCR assays only amplified between 5-20% of the samples after the secondary PCR
259
amplification (Fig 2).
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Reverse line blot hybridization and nucleotide sequence analysis. A reverse line blot
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hybridization assay was used for high-through put identification of 23S-5S nested amplicons
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from D. variabilis ticks. All control DNAs hybridized with the Rickettsia genus probe (GP-RICK)
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and also hybridized with the intended DNA probes specific for SFG and TG species of
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Rickettsia. All 18 of the 23S-5S positive samples from 40 D. variabilis samples hybridized to
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Rickettsia genus-specific probe. The samples predominantly hybridized to the “R. amblyommii”
266
specific probe, but also to probes for R. conorii, R. montanensis, R. bellii, and R. massiliae.
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Some unknown species of Rickettsia and SFG species were also detected (Table 2). The RLB
268
results were further evaluated by nucleotide sequencing of 23S-5S amplicons (Table 2). Blast
269
results of the 23S-5S sequences showed high (99-100%) similarity to “R. amblyommii”, R.
270
montanensis, R. bellii, R. felis, and the presence of some potentially novel Rickettsia species.
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Nucleotide sequence analysis was generally congruent with RLB hybridization results (Table 2).
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Concurrently, amplicons from the other PCR targets (gltA, ompA and 17-kDA) that produced
273
visual bands on agarose gels were also sequenced and the sequences showed highest
274
similarity to R. montanensis and “R. amblyommii”. 11
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Phylogenetic analysis. Phylogenetic analysis of Rickettsia spp. using neighbor joining
276
method showed that Dermacentor variabilis ticks contained a diverse group of rickettsiae.
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Potentially novel Rickettsia spp. with 97% similarity to R. bellii, were placed on a separate
278
branch. Multiple ticks contained a Rickettsia species that was placed on the same branch as R.
279
conorii. Rickettsiae identified through sequence match analysis as “R. amblyommii”, R.
280
montanensis and R. felis in this study were aligned with these species in the phylogenetic tree
281
(Fig. 3).
282
DISCUSSION
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Our novel nested 23S-5S IGS PCR assay was shown to be a robust molecular tool for detecting
284
Rickettsia species. Developing new and improving the existing diagnostic tools facilitates
285
molecular epidemiological investigations of tick-borne diseases (17). In this regard, the new
286
nested assay should find utility in detecting Rickettsia spp. in ticks and clinical samples.
287
Pathogen surveillance in field-collected ticks by PCR amplifying specific gene targets is a well-
288
adopted practice for determining the prevalence and diversity of the genus Rickettsia in a given
289
environment (8, 19-21, 24, 41). We found that our nested 23S-5S IGS PCR assay out
290
performed 17-kDA, ompA and gltA that are commonly used gene targets in molecular surveys
291
of rickettsiae in ticks and other environmental samples. In the light of the increasing number of
292
cases of SFG rickettsiosis (4), which could potentially be caused by new Rickettsia spp.(4, 6), it
293
is essential to develop new and/or improve existing molecular assays to increase the detection
294
rate of SFG and other rickettsiae vectored by ticks and other blood feeding arthropods (17).
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The new nested 23S-5S IGS assay amplified all 11 positive control Rickettsia spp.
296
representing SFG, TG and ancestral groups. In contrast, at the same template gDNA
297
concentration, other gene targets failed to amplify all phylogenetic groups of Rickettsia, making
298
23S-5S IGS a robust target for Pan-Rickettsia detection. The utility of a conventional PCR
299
assay targeting Rickettsia 23S-5S IGS was reported previously (22); however, adding a nested
12
300
step substantially improved the sensitivity of the assay. The highly conserved ends and the
301
hypervariable central regions make it an ideal target for understanding the phylogenetic
302
relationships of SFG and other species of Rickettsia (2, 25). The greater efficiency of 23S-5S
303
IGS for Rickettsia detection compared to other PCR targets is likely due to a higher overall
304
coverage and primer specificity across the genus. Occurrence of 23S-5S IGS in SFG, typhus
305
and ancestral rickettsial groups is a major strength of our newly developed assay.
306
The American dog tick (D. variabilis) is an established vector of RMSF. However, recent
307
studies have generally reported that
